Francisella tularensis, the causative agent of the zoonotic disease tularemia, is a highly infectious facultative intracellular pathogen. F. tularensis is classified by the United States Center for Disease Control and Prevention as a category A agent, which exhibits high mortality rate and is a potential bioterrorism agent. The recent availability of genomic information for F. tularensis subspecies revealed that the genome encodes two paralogous copies of the rpoA gene, which encode non-identical RNA polymerase (RNAP) ? subunits. RNAP is an evolutionary conserved enzyme responsible for all transcription in bacterial cells. In all bacteria studied to date, a dimer of identical ? subunits (?2) initiates the assembly of the catalytically proficient RNAP core (subunit composition ?2??'). The unprecedented presence of two genes encoding different RNAP ? subunits means that in F. tularensis four different ? dimers (?1)2, ?1?2, ?2?1, and (?2)2 may form, leading to the presence of four distinct RNAP core species in the same cell. Because ? subunits, through their C-terminal domains, participate in promoter recognition by sequence-specific protein-DNA interaction with promoter UP elements and interact with a large set of transcription activation proteins, the presence of two different ? subunits may have a profound effect on the regulation of expression of genes in F. tularensis. The main goal of this proposal is to determine the roles played by non-identical ? subunits in regulation of gene expression in F. tularensis. For this purpose, we are going to elucidate the role of each ? subunit in the RNAP assembly, promoter recognition, and transcriptional activation (repression). To this end, recombinant versions of all possible F. tularensis RNAP core enzymes will be prepared by heterologous co-overexpression in E. coli or by in vitro assembly, corresponding RNAP holoenzymes will be prepared and their properties (promoter specificity, transcription elongation and termination properties and ability to respond to transcription regulators) will be established using a set of discriminative in vitro transcription assays. In addition to general significance for the understanding of basic mechanisms of RNAP function and regulation in bacteria, the proposed studies of the highly unusual F. tularensis RNAP and its variants of different subunit composition may lead to development of novel inhibitors of F. tularensis transcription and antibacterial agents. PUBLIC HEALTH RELEVANCE: Francisella tularensis, the causative agent of the zoonotic disease tularemia, a highly infectious facultative intracellular pathogen, possesses unusual RNA polymerase (RNAP), which contains two different alpha subunits. Because of important role of alpha subunit in RNAP assembly, promoter recognition and gene regulation, systematic study of RNAP from this pathogenic bacterium will uncover a novel mechanism of transcription regulation and may lead to development of new transcription inhibitors of F. tularensis and antibacterial agents.